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Spatial memory is responsible for encoding spatial information to form a path, storing this mental representation, and evaluating and recovering spatial configurations to find a target location in the environment. It is mainly supported by the hippocampus and its interaction with other structures, such as the prefrontal cortex, and emerges in rodents around postnatal day (PND) 20. Sex differences in spatial tasks have been found in adults, with a supposedly better performance in males. However, few studies have examined sex differences in orientation throughout postnatal development. This study aimed to analyse the performance of juvenile (PND 23) male (n = 18) and female (n = 21) Wistar rats in a spatial reference memory task in the Morris water maze (MWM) with two different training regimes in the acquisition phase, and their subjacent metabolic brain activity. Based on sex, subjects were assigned to two different groups: one that performed four learning trials per day (n = 9 males and n = 8 females) and the other that was submitted to two trials per day (n = 9 males and n = 13 females). After the behavioural protocols, metabolic activity was evaluated using cytochrome c oxidase histochemistry. Results showed no metabolic brain or behavioural differences in the four-trial protocol performance, in which both sexes reached the learning criterion on the fourth day. By contrast, the two-trial protocol revealed an advantage for females, who reached the learning criterion on day four, whereas males needed more training and succeeded on day six. The female group showed lower metabolic activity than the male group in the cingulate and prelimbic cortex. These results suggest a faster consolidation process in the female group than the male group. Further research is needed to understand sex differences in spatial memory at early stages., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest to disclose., (Copyright © 2023. Published by Elsevier Inc.)

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation technique that is able to generate causal-based interferences between brain networks and cognitive or behavioral responses. It has been used to improve cognition in several disease models. However, although its exploration in healthy animals is essential to attribute its pure effect in learning and memory processes, studies in this regard are scarce. We aimed to evaluate whether rTMS leads to memory facilitation in healthy rats, and to explore the brain-related oxidative metabolism. We stimulated healthy Wistar rats with a high-frequency (100 Hz) and low-intensity (0.33 T) protocol during three consecutive days and evaluated the effect on the performance of an allocentric spatial reference learning and memory task. Following the last day of learning, we assessed oxidative brain metabolism through quantitative cytochrome c oxidase (CCO) histochemistry. The results showed that rTMS did not improve spatial memory in healthy rats, but the behavioral outcome was accompanied by a CCO reduction in the prefrontal, retrosplenial, parietal, and rhinal cortices, as well as in the striatum, amygdala, septum, mammillary bodies, and the hippocampus, reflecting a lower metabolic activity. In conclusion, rTMS induces a highly efficient use of brain regions associated with spatial memory., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Photobiomodulation (PBM) or the use of red to near-infrared irradiation spectrum, is a non-invasive intervention that produces neurostimulatory effects and reaches benefits in several pathologies as well as in healthy subjects. The main objective of this study was to evaluate and compare the effects of PBM in a rat brain network on basal state and functional activity during the execution of a reversal task. Twenty-eight rats were divided into four groups: control group (n = 7), control photobiomodulation group (n = 8), behavioural group (n = 6) and behavioural photobiomodulation group (n = 7). Reversal memory was assessed using a Morris water maze and cytochrome c oxidase (CCO) was used as a brain metabolic activity marker. After five days of PBM, the control photobiomodulation group showed a decrease of CCO levels in the striatum, medial septum, entorhinal, hippocampus, amygdala, thalamus, mammillary nuclei and VTA. Both behavioural groups performed the task correctly, however, the behavioural photobiomodulation group displayed CCO reduction in some regions involved in the execution of the reversal task: septum, entorhinal, CA1, CA3, central amygdala and supramammilar, along with higher levels in accumbens. These results could show the effect of PBM on active brain networks. Further studies will be necessary to elucidate its effects in different brain networks that are involved in the execution of other memory tasks., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Spatial orientation is a cognitive ability that is indispensable for survival. Several visual distal cues present in the context can be integrated, establishing a cognitive map. Although there is cumulative evidence about the neural substrate involved in spatial memory acquisition, the brain networks mediating the processes involved in the retrieval of allocentric spatial memories have been studied less. Here, we aimed to explore the role of neuronal oxidative metabolism in the retrieval of allocentric spatial memories through cytochrome c oxidase (CCO) histochemistry seven, 15, 30, 45, and 60 days after task acquisition. Our behavioural results show that spatial memory retrieval in male and female rats is preserved seven, 15, and 30 days post-acquisition, but there is forgetfulness after this time, with subjects not being able to remember the position of the hidden platform after 45 and 60 dfearays. Regarding the study of male brain metabolism, we observed reduced CCO activity in the medial prefrontal cortex, the parietal, retrosplenial, rhinal cortex, and the hippocampal regions in all the groups that failed to solve the task. Similar results were found for female brain oxidative metabolism, in addition to certain differences between succefearssful-retrieval female groups. In conclusion, our work adds information about the behavioural retrieval of an allocentric spatial reference task, suggesting that recovering spatial information seven, 15, and 30days after acquisition is a simple task that does not require a high metabolic demand, in both male and female rats., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Early life stress is associated with long-term and pervasive adverse effects on neuroendocrine development, affecting normal cognitive and emotional development. Experimental manipulations like environmental enrichment (EE) may potentially reverse the effects of early life stress induced by maternal separation (MS) paradigm in rodents. However, the functional brain networks involved in the effects of EE after prolonged exposure to MS have not yet been investigated. In order to evaluate possible changes in brain functional connectivity induced by EE after MS, quantitative cytochrome c oxidase (CCO) histochemistry was applied to determine regional brain oxidative metabolism in adult male rats. Unexpectedly, results show that prolonged MS during the entire weaning period did not cause any detrimental effects on spatial learning and memory, including depressive-like behavior evaluated in the forced-swim test, and decreased anxiety-like behavior. However, EE seemed to alter anxiety- and depression-like behaviors in both control and MS groups, but improved spatial memory in the latter groups. Analysis of brain CCO activity showed significantly lower metabolic capacity in most brain regions selected in EE groups probably associated with chronic stress, but no effects of MS on brain metabolic capacity. In addition, principal component analysis of CCO activity revealed increased large-scale functional brain connectivity comprising at least three main networks affected by EE in both MS and control groups. Moreover, EE induced a pattern of functional brain connectivity associated with stress and anxiety-like behavior as compared with non-enriched groups. In conclusion, EE had differential effects on cognition and emotional behavior irrespective of exposure to MS. In view of the remarkable effects of EE on brain function and behavior, implementation of rodent housing conditions should be optimized by evaluating the balance between scientific validity and animal welfare., Competing Interests: The authors have declared that no competing interests exist.

Non-alcoholic steatohepatitis (NASH) is one of the most prevalent diseases worldwide. While it has been suggested to cause nervous impairment, its neurophysiological basis remains unknown. Therefore, the aim of this study is to unravel the effects of NASH, through the interrelationship of liver, gut microbiota, and nervous system, on the brain and human behavior. To this end, 40 Sprague-Dawley rats were divided into a control group that received normal chow and a NASH group that received a high-fat, high-cholesterol diet. Our results show that 14 weeks of the high-fat, high-cholesterol diet induced clinical conditions such as NASH, including steatosis and increased levels of ammonia. Rats in the NASH group also demonstrated evidence of gut dysbiosis and decreased levels of short-chain fatty acids in the gut. This may explain the deficits in cognitive ability observed in the NASH group, including their depressive-like behavior and short-term memory impairment characterized in part by deficits in social recognition and prefrontal cortex-dependent spatial working memory. We also reported the impact of this NASH-like condition on metabolic and functional processes. Brain tissue demonstrated lower levels of metabolic brain activity in the prefrontal cortex, thalamus, hippocampus, amygdala, and mammillary bodies, accompanied by a decrease in dopamine levels in the prefrontal cortex and cerebellum and a decrease in noradrenalin in the striatum. In this article, we emphasize the important role of ammonia and gut-derived bacterial toxins in liver-gut-brain neurodegeneration and discuss the metabolic and functional brain regional deficits and behavioral impairments in NASH., Competing Interests: The authors have declared that no competing interests exist.

Social isolation during adulthood is a frequent problem that leads to a large variety of adverse emotional and cognitive effects. However, most of the social isolation rodent procedures begin the separation early post-weaning. This work explores locomotor activity, anxiety-like behaviour, and spatial working memory after twelve weeks of adult social isolation. In order to study the functional contribution of selected brain areas following a working memory task, we assessed neuronal metabolic activity through quantitative cytochrome oxidase histochemistry and c-Fos immunohistochemistry. Behaviourally, we found that isolated animals (IS) showed anxiety-like behaviour and worse working memory than controls, whereas motor functions were preserved. Moreover, IS rats showed lower levels of learning-related c-Fos immunoreactivity, compared to controls, in the medial prefrontal cortex (mPFC), ventral tegmental area (VTA), and nucleus accumbens shell. In addition, the IS group showed lower neuronal metabolic activity in the mPFC, VTA, and CA1 subfield of the hippocampus. These results indicate that twelve weeks of social isolation in adult rats leads to different behavioural and brain alterations, and they highlight the importance of social support, not only in development, but also in adulthood., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Exposure to maternal separation (MS) in rodents may have long-lasting consequences for the structure and function of several brain regions, eventually associated with alterations in cognition and emotion later in life. Post-weaning environmental enrichment (EE) has been reported to ameliorate the detrimental effects of exposure to early life stress mainly in the hippocampus., Method: In vivo magnetic resonance imaging (MRI) was applied to evaluate possible volumetric changes in the dorsal and ventral hippocampus, the medial prefrontal cortex and the dorsal striatum of 90-day-old male rats after daily MS for 240 min from postnatal days 2-21., Results: No significant volume changes were found in the selected brain regions in MS animals as compared with an age-matched control group. However, additional groups of control and MS animals with EE from days 21-60 showed significant volume increases in the medial prefrontal cortex and the ventral hippocampus as compared to the groups without EE. In addition, general hemispheric asymmetry was found in the volume of the brain regions measured., Conclusions: Our results demonstrate that EE could have differential effects depending on previous exposure to MS and on the development of brain lateralization.

Prenatal and perinatal alcohol exposure caused by maternal alcohol intake during gestation and lactation periods can have long-lasting detrimental effects on the brain development and behaviour of offspring. Children diagnosed with Foetal Alcohol Spectrum Disorders (FASD) display a wide range of cognitive, emotional and motor deficits, together with characteristic morphological abnormalities. Maternal alcohol binge drinking is particularly harmful for foetal and early postnatal brain development, as it involves exposure to high levels of alcohol over short periods of time. However, little is known about the long-term effects of maternal alcohol binge drinking on brain function and behaviour. To address this issue, we used pregnant C57BL/6 female mice with time-limited access to a 20% v/v alcohol solution as a procedure to model alcohol binge drinking during gestation and lactational periods. Male offspring were behaviourally tested during adolescence (30 days) and adulthood (60 days), and baseline neural metabolic capacity of brain regions sensitive to alcohol effects were also evaluated in adult animals from both groups. Our results show that prenatal and postnatal alcohol exposure caused age-dependent changes in spontaneous locomotor activity, increased anxiety-like behaviour and attenuated alcohol-induced conditioned place preference in adults. Also, significant changes in neural metabolic capacity using cytochrome c oxidase (CCO) quantitative histochemistry were found in the hippocampal dentate gyrus, the mammillary bodies, the ventral tegmental area, the lateral habenula and the central lobules of the cerebellum in adult mice with prenatal and postnatal alcohol exposure. In addition, the analysis of interregional CCO activity correlations in alcohol-exposed adult mice showed disrupted functional brain connectivity involving the limbic, brainstem, and cerebellar regions. Finally, increased neurogenesis was found in the dentate gyrus of the hippocampus of alcohol-exposed offspring, suggesting neuroadaptive effects due to early alcohol exposure. Our results demonstrate that maternal binge-like alcohol drinking causes long-lasting effects on motor and emotional-related behaviours associated with impaired neuronal metabolic capacity and altered functional brain connectivity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Early life stress comprises a wide range of adverse events that can occur in the subject's early developmental stages: from child abuse to rodent repeated maternal separation models. This study used young adult male Wistar rats that were maternally raised (AFR), maternally separated from post-natal day (PND) 1 to PND10 (MS10), or maternally separated from PND1 to PND21 (MS21), to assess the effects of maternal separation on spatial learning and memory using the Morris Water Maze (MWM). We also performed quantitative cytochrome oxidase (COx) histochemistry on some selected brain areas in order to find out whether maternal separation affects brain energy metabolism. We obtained a similar spatial learning pattern in maternally raised and maternally separated subjects, with MS10 showing faster acquisition; however, different brain networks with different energy consumptions were used by each group. MS10 spent more energy to get the same behavioral outputs, whereas MS21 resembled AFR more in term of energy demands., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background & Aims: Minimal hepatic encephalopathy (mHE) has been shown to affect daily functioning, quality of life, driving and overall mortality. However, little is known about treating or diagnosing early impairments in mHE. We studied one of its precipitating factors, portal hypertension which is driving the inflammatory process behind mHE. The purpose was to describe an indirect diagnostic method able to detect the pathology at early stages based on the study of the vascularization and mast cells conjunctival hyperplasia as secondary inflammatory response associated to portal hypertension. Finally, we correlated the presence of histological changes in the eye in mHE with deficits in behavioral task acquisition., Methods: Rats were trained on a stimulus-response task and a spatial working memory task using the Morris water maze. Two groups of animals were used: a SHAM (sham-operated) group (n=10) and a portal hypertension (HT) group (n=10). The triple portal vein ligation method was used to create an animal model of mHE. Latencies to reach the platform, number of glial fibrillary acidic protein-immunoreactive astrocytes (GFAP-IR), mast cell expression and presence/absence of blood and lymphatic vessels were examined., Results: There were differences in stimulus-response behavioral performance, with a deficit in the acquisition in the HT group. However, no differences between groups were found on the spatial working memory task. At the same time, differences between groups were found in the GFAP-IR density, which was lower in the HT group, and in the number of mast cells and the presence of vessels, which were higher in the HT group., Conclusions: In this study, we provide the first preliminary insight into the validity of exploring the eye as a possible tool to assess the diagnosis of mHE conditions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Minimal hepatic encephalopathy (MHE) has been shown to affect daily functioning, quality of life, driving and overall mortality. However, little is known about treating or diagnosing early impairments involved in MHE. We studied one of its precipitating factors, portal hypertension. The purpose was to evaluate an enhancement in neuronal metabolism through low-light-level therapy (LLLT) and whether this therapy has effects on behavioural task acquisition. Rats were trained to perform a stimulus-response task using the Morris water maze. Three groups of animals were used: a SHAM (sham-operated) group (n = 7), a portal hypertension (PH) group (n = 7) and a PH + LLLT group (n = 7). The triple portal vein ligation method was used to create an animal model of the early developmental phase of HE, and then the animals were exposed to 670 + 10 nm LED light at a dose of 9 J/cm 2 once a day for 7 days. The metabolic activity of the brains was studied with cytochrome c oxidase histochemistry. There were differences in behavioural performance, with an improvement in the PH + LLLT group. Energetic brain metabolism revealed significant differences between the groups in all the brain structures analysed, except the anterodorsal thalamus. At the same time, in different brain networks, the PH group showed a more complicated relationship among the structures, while the SHAM and PH + LLLT groups had similar patterns. In this study, we provide the first preliminary insights into the validity of LLLT as a possible intervention to improve memory under minimal hepatic encephalopathy conditions.

Mastering the Morris water maze (MWM) requires the animal to consolidate, retain and retrieve spatial localizations of relevant visual cues. However, it is necessary to investigate whether a reorganization of the neural networks takes place when part of the spatial information is removed. We conducted four experiments using the MWM. A classical reference memory procedure was performed over five training days, RM5 (n=7), and eight days, RM8 (n=7), with the whole room and all the spatial cues presented. Another group of animals were trained in the same protocol, but they received an additional day of training with only partial cues, PC (n=8). Finally, a third group of animals performed the classical task, followed by an overtraining with partial cues for four more days, OPC (n=8). After completing these tasks, cytochrome c-oxidase activity (CO) in several brain limbic system structures was compared between groups. In addition, c-Fos positive cells were measured in the RM5, RM8, PC and OPC groups. No significant differences were found among the four groups in escape latencies or time spent in the target quadrant. CO revealed involvement of the prefrontal and parietal cortices, dorsal and ventral striatum, CA1 and CA3 subfields of the dorsal hippocampus, basolateral and lateral amygdala, and mammillary nuclei in the PC group, compared to the RM group. In the OPC group, involvement of the ventral striatum and anteroventral thalamus and the absence of amygdala involvement were revealed, compared to the PC group. C-Fos results highlighted the role of the prefrontal cortex, dorsal striatum, anterodorsal thalamus and CA3 in the PC group, compared to the OPC, RM5 and RM8 groups. The animals were able to find the escape platform even when only a portion of the space where the cues were placed was available. Although the groups did not differ behaviorally, energetic brain metabolism and immediate early gene expression revealed the engagement of different neural structures in the groups that received more training without the entire surrounding space., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Hepatic encephalopathy (HE) is a neurological complication observed in patients with liver disease. Patients who suffer from HE present neuropsychiatric, neuromuscular and behavioral symptoms. Animal models proposed to study HE resulting from cirrhosis mimic the clinical characteristics of cirrhosis and portal hypertension, and require the administration of hepatotoxins such as thioacetamide (TAA). The aim of this study was to assess the effects of a high protein diet on motor function, anxiety and memory processes in a model of cirrhosis induced by TAA administration. In addition, we used cytochrome c-oxidase (COx) histochemistry to assess the metabolic activity of the limbic system regions. Male rats were distributed into groups: control, animals with cirrhosis, Control rats receiving a high protein diet, and animals with cirrhosis receiving a high protein diet. Results showed preserved motor function and normal anxiety levels in all the groups. The animals with cirrhosis showed an impairment in active avoidance behavior and spatial memory, regardless of the diet they received. However, the animals with cirrhosis and a high protein diet showed longer escape latencies on the spatial memory task. The model of cirrhosis presented an under-activation of the dentate gyrus and CA3 hippocampal subfields and the medial part of the medial mammillary nucleus. The results suggest that a high protein intake worsens spatial memory deficits shown by the TAA-induced model of cirrhosis. However, high protein ingestion has no influence on the COx hypoactivity associated with the model., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although often not considered clinically relevant and, therefore, not diagnosed or treated, minimal hepatic encephalopathy (MHE) has been shown to affect daily functioning, quality of life, driving and overall mortality. To discover early impairments involved in MHE, we studied one of its precipitating factors, portal hypertension. Rats were trained on a stimulus-response task using the Morris water maze. Two groups of animals were used: a SHAM (sham-operated) group (n= 13) and a portal hypertension (PH) group (n= 13). The triple portal vein ligation method was used to create an animal model of an early developmental phase of HE. Brain metabolic activity was studied with cytochrome c-oxidase histochemistry (C.O.). Neuronal nuclear volume was assessed by nucleator probe; the number of glial fibrillary acidic protein-immunoreactive astrocytes (GFAP-IR) and proinflammatory mediators was measured. The results revealed that the PH group was not able to reach the behavioural criterion, in contrast to the SHAM group. The metabolic brain consumption revealed decreased C.O. activity in the ventral striatum. The PH group showed lower density of GFAP-IR and an increase in the tumour necrotic factor-α (TNF-α). The PH group showed decreased neuronal nuclear volume in the dorsal striatum. On the contrary, increased neuronal nuclear volume was found in the ventral striatum. For the first time, a relationship has been established between inflammation, astrocytic and neural damage, and brain metabolic impairment in a model of MHE. Disruption of the striatum and related structures was highlighted as the main target in early stages of HE. Finally, a simple task was presented to assess the subtle impairments found in the clinic, which could provide fresh insights into the development of new tools for the assessment of MHE.

Visual discrimination tasks have been widely used to evaluate many types of learning and memory processes. However, little is known about the brain regions involved at different stages of visual discrimination learning. We used cytochrome c oxidase histochemistry to evaluate changes in regional brain oxidative metabolism during visual discrimination learning in a water-T maze at different time points during training. As compared with control groups, the results of the present study reveal the gradual activation of cortical (prefrontal and temporal cortices) and subcortical brain regions (including the striatum and the hippocampus) associated to the mastery of a simple visual discrimination task. On the other hand, the brain regions involved and their functional interactions changed progressively over days of training. Regions associated with novelty, emotion, visuo-spatial orientation and motor aspects of the behavioral task seem to be relevant during the earlier phase of training, whereas a brain network comprising the prefrontal cortex was found along the whole learning process. This study highlights the relevance of functional interactions among brain regions to investigate learning and memory processes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The present study investigated the behavioural performance and the contributions of different brain regions on a spatial task performed by Wistar rats in the Cincinnati water maze (CWM) in two conditions: one where both distal and proximal visual cues were available (CWM-light group, n=7) and another where visual cues were eliminated by testing in complete darkness (CWM-dark group, n=7). There were differences in the behavioural performance. Energetic brain metabolism revealed significant differences in the infralimbic, orbitofrontal cortex and anterodorsal striatum. At the same time different brain networks were found. The CWM-light group showed a relationship between the orbitofrontal cortex and medial septum, whereas the CWM-dark group revealed three different networks involving the prefrontal cortex, ventral striatum, hippocampus and amygdala nuclei. The study shows that brain activation differs in these two conditions., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Patients with minimal hepatic encephalopathy exhibit early impairments in their ability to shift attentional set. We employed a task-switching protocol to evaluate brain network changes. Strategy switching requires the modification of both the relevant stimulus dimension and the required memory system. Rats were trained in an allocentric (A) and a cue-guided (C) task using a four-arm maze. To examine priming, we changed the order in which the tasks were presented. Five groups of animals were used: a SHAM (sham-operated) A-C group (n=10), a SHAM C-A group (n=8), a PH (portal hypertension) A-C group (n=8), PH C-A group (n=8), and a naïve group (n=10). The triple portal vein ligation method was used to create an animal model of the early evolutive phase of PH. The animals were tested in the four-arm radial water maze in a single 10-trial session each day for six days (three days for the allocentric task and three days for the cue-guided task). The metabolic activities of the brains were studied with cytochrome oxidase histochemistry, and brain network changes were assessed with principal component analysis. The behavioural results revealed significant increases in the numbers of correct choices across training days in all groups studied, and facilitation of the acquisition of the second task was present in the C-A groups. Moreover, different brain network activities were found; in the experimental groups, the performance of A-C switch involved the prefrontal cortex, and the key structures involved in the C-A switch in the other groups were the dentate gyrus of the dorsal hippocampus and the basolateral and central amygdala. These networks have a common nucleus of structures (i.e., the parietal cortex and the dorsal and ventral striatum), whereas other structures were specifically involved in each type of strategy, suggesting that these regions are part of both circuits and may interact with one another during learning., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ammonia is thought to be central in the development of hepatic encephalopathy. However, the specific relation of ammonia with brain energy depletions and learning has not been studied. Our work attempts to reproduce an increase in rat cerebral ammonia level, study the hyperamonemic animals' performance of two learning tasks, an allocentric (ALLO) and a cue guided (CG) task, and elucidate the contribution of hyperammonemia to the differential energy requirements of the brain limbic system regions involved in these tasks. To assess these goals, four groups of animals were used: a control (CHA) CG group (n = 10), a CHA ALLO group (n = 9), a hyperammonemia (HA) CG group (n = 7), and HA ALLO group (n = 8). Oxidative metabolism of the target brain regions were assessed by histochemical labelling of cytochrome oxidase (C.O.). The behavioural results revealed that the hyperammonemic rats were not able to reach the behavioural criterion in either of the two tasks, in contrast to the CHA groups. The metabolic brain consumption revealed increased C.O. activity in the anterodorsal thalamus when comparing the HA ALLO group with the CHA ALLO group. Significant differences between animals trained in the CG task were observed in the prelimbic, infralimbic, parietal, entorhinal and perirhinal cortices, the anterolateral and anteromedial striatum, and the basolateral and central amygdala. Our findings may provide fresh insights to reveal how the differential damage to the brain limbic structures involved in these tasks differs according to the degree of task difficulty.

While the neuronal basis of spatial memory consolidation has been thoroughly studied, the substrates mediating the process of extinction remain largely unknown. This study aimed to evaluate the functional contribution of selected brain regions during the extinction of a previously acquired spatial memory task in the Morris water maze. For that purpose, we used adult male Wistar rats trained in a spatial reference memory task. Learning-related changes in c-Fos inmunoreactive cells after training were evaluated in cortical and subcortical regions. Results show that removal of the hidden platform in the water maze induced extinction of the previously reinforced escape behavior after 16 trials, without spontaneous recovery 24h later. Extinction was related with significantly higher numbers of c-Fos positive nuclei in amygdala nuclei and prefrontal cortex. On the other hand, the lateral mammillary bodies showed higher number of c-Fos positive cells than the control group. Therefore, in contrast with the results obtained in studies of classical conditioning, we show the involvement of diencephalic structures mediating this kind of learning. In summary, our findings suggest that medial prefrontal cortex, the amygdala complex and diencephalic structures like the lateral mammillary nuclei are relevant for the extinction of spatial memory., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Several studies have reported the brain regions involved in response learning. However, there is discrepancy regarding the lighting conditions in the experimental setting (i.e. under dark or light conditions). In this regard, it would be relevant to know if the presence/absence of visual cues in the environment has any effect in the brain networks involved in a response learning task. Animals were trained in a water T-maze under two different lighting conditions (light versus dark). All subjects reached the learning criterion of 80% correct arm choices. Quantitative cytochrome oxidase (CO) histochemistry was used as a metabolic brain mapping technique. Our results show that the ventral hippocampus and the parietal cortex are associated with the acquisition of a response learning task regardless of lighting conditions. In addition, when the same task is run in the dark, widespread recruitment of structures involving cortical, limbic and striatal regions was found., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

We assessed the effect of 3h of environmental enrichment (EE) exposure per day started at different ages (3 and 18months old) on the performance in a spatial memory task and on brain regions involved in the spatial learning (SPL) process using the principal component analysis (PCA). The animals were tested in the four-arm radial water maze (4-RAWM) for 4days, with six daily trials. We used cytochrome c oxidase (COx) histochemistry to determine the brain oxidative metabolic changes related to age, SPL and EE. Behavioural results showed that the enriched groups, regardless of their age, achieved better performance in the spatial task. Interestingly, in the case of the distance travelled in the 4-RAWM, the effect of the EE was dependent on the age, so the young enriched group travelled a shorter distance compared to the aged enriched group. Respect to COx histochemistry results, we found that different brain mechanisms are triggered in aged rats to solve the spatial task, compared to young rats. PCA revealed the same brain functional network in both age groups, but the contribution of the brain regions involved in this network was slightly different depending on the age of the rats. Thus, in the aged group, brain regions involved in anxiety-like behaviour, such as the amygdala or the bed nucleus of the stria terminalis had more relevance; whereas in the young enriched group the frontal and the hippocampal subregions had more contribution., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The involvement of different brain regions in place- and response-learning was examined using a water cross-maze. Rats were trained to find the goal from the initial arm by turning left at the choice point (egocentric strategy) or by using environmental cues (allocentric strategy). Although different strategies were required, the same maze and learning conditions were used. Using cytochrome oxidase histochemistry as a marker of cellular activity, the function of the 13 diverse cortical and subcortical regions was assessed in rats performing these two tasks. Our results show that allocentric learning depends on the recruitment of a large functional network, which includes the hippocampal CA3, dentate gyrus, medial mammillary nucleus and supramammillary nucleus. Along with the striatum, these last three structures are also related to egocentric spatial learning. The present study provides evidence for the contribution of these regions to spatial navigation and supports a possible functional interaction between the two memory systems, as their structural convergence may facilitate functional cooperation in the behaviours guided by more than one strategy. In summary, it can be argued that spatial learning is based on dynamic functional systems in which the interaction of brain regions is modulated by task requirements., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Our aim was to assess the way that middle-aged rats solve spatial learning tasks that can be performed using different strategies. We assessed the brain networks involved in these spatial learning processes using Principal Component Analysis. Two tasks were performed in a complex context, a four-arm radial maze, in which each group must use either an allocentric or an egocentric strategy. Another task was performed in a simple T-maze in which rats must use an egocentric strategy. Brain metabolic activity was quantified to evaluate neural changes related to spatial learning in the described tasks. Our findings revealed that two functional networks are involved in spatial learning in aged rats. One of the networks, spatial processing, is composed of brain regions involved in the integration of sensory and motivational information. The other network, context-dependent processing, mainly involves the dorsal hippocampus and is related to the processing of contextual information from the environment. Both networks work together to solve spatial tasks in a complex spatial environment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The specific brain regions and circuits involved in the acquisition and expression of contextual fear conditioning are still a matter of debate. To address this issue, regional changes in brain metabolic capacity were mapped during the acquisition and expression of contextual fear conditioning using cytochrome oxidase (CO) quantitative histochemistry. In comparison with a group briefly exposed to a conditioning chamber, rats that received a series of randomly presented footshocks in the same conditioning chamber (fear acquisition group) showed increased CO activity in anxiety-related brain regions like the ventral periaqueductal gray, the ventral hippocampus, the lateral habenula, the mammillary bodies, and the laterodorsal thalamic nucleus. Another group received randomly presented footshocks, and it was re-exposed to the same conditioning chamber one week later (fear expression group). The conditioned group had significantly higher CO activity as compared with the matched control group in the following brain regions: the ventral periaqueductal gray, the central and lateral nuclei of the amygdala, and the bed nucleus of the stria terminalis. In addition, analysis of functional brain networks using interregional CO activity correlations revealed different patterns of functional connectivity between fear acquisition and fear expression groups. In particular, a network comprising the ventral hippocampus and amygdala nuclei was found in the fear acquisition group, whereas a closed reciprocal dorsal hippocampal network was detected in the fear expression group. These results suggest that contextual fear acquisition and expression differ as regards to the brain networks involved, although they share common brain regions involved in fear, anxiety, and defensive behavior., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Hepatic encephalopathy is a syndrome whose physiopathology is poorly understood; therefore, current diagnostic tests are imperfect and modern therapy is nonspecific. Particularly, it has been suggested that inflammation plays an important role in the pathogenesis of portal hypertensive encephalopathy in the rat., Aim: We have studied an experimental model of portal hypertension based on a triple partial portal vein ligation in the rat to verify this hypothesis., Methods: One month after portal hypertension we assayed in the splanchnic area (liver, small bowel and mesenteric lymph nodes) and in the central nervous system (hippocampus and cerebellum) fractalkine (CX3CL1) and stromal cell-derived factor alpha (SDF1-α) as well as their respective receptors (CX3CR1 and CXCR4) because of their key role in inflammatory processes., Results: The significant increase of fractalkine in mesenteric lymph nodes (P<0.05) and its receptor (CX3CR1) in the small bowel (P<0.05) and hippocampus (P<0.01), associated with the increased expression of SDF1-α in the hippocampus (P<0.01) and the cerebellum (P<0.01) suggest that prehepatic portal hypertension in the rat induces important alterations in the expression of chemokines in the gut-brain axis., Conclusion: The present study revealed that portal hypertension is associated with splanchnic-brain inflammatory alterations mediated by chemokines.

Patients with liver malfunction often suffer from hepatic encephalopathy, a neurological complication which can affect attention and cognition. Diverse experimental models have been used to study brain alterations that may be responsible for hepatic encephalopathy symptoms. The aim of the study was to determine whether cognitive impairment found in cirrhosis could be due to disturbance of acetylcholinesterase activity. Acetylcholinesterase activity was assessed in the brains of Wistar rats with thioacetamide-induced cirrhosis. The cirrhotic group displayed up-regulation of acetylcholinesterase levels in the entorrhinal cortex, anterodorsal and anteroventral thalamus and accumbens, whereas down-regulation was found in the CA1, CA3 and dentate gyrus of the hippocampus. Our results indicate that the experimental model of hepatic encephalopathy by chronic administration of thioacetamide presents alterations of acetylcholinesterase activity in brain limbic system regions, which play a role in attention and memory., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

We evaluated change in cytochrome oxidase (COx) activity of the hippocampus and related structures of the limbic system following spatial working memory learning in rats after treatment with tacrine (8.0mg/kg). Control groups treated with saline and tacrine and an untreated group were added. Acetylcholinesterase optical density levels were also measured. The tacrine and saline groups showed similar behavioral results, but a decrease in COx activity was found in the tacrine group in the prefrontal cortex, nucleus accumbens, anterior thalamus, hippocampus and nucleus basalis of Meynert. Similarly, acetylcholinesterase levels of the tacrine group were lower in most of the regions. Learning-related increase in COx activity was found in the prefrontal cortex and dentate gyrus in the saline group. The tacrine group presented the same increase in the anterodorsal thalamus, dentate gyrus, CA3 and mammillary nuclei. These results suggest that inhibition of the acetylcholinesterase produces a different pattern of learning-related neuronal activity in the limbic system of the rat.

Background & Aims: Gold nanoparticles (GNPs) have promising applications for drug delivery as well as for the diagnosis and treatment of several pathologies, such as those related to the CNS. However, GNPs are retained in a number of organs, such as the liver and spleen. Owing to their negative charge and/or processes of opsonization, GNPs are retained by the reticuloendothelial system, thereby decreasing their delivery to the brain. It is therefore crucial to modify the nanoparticle surface in order to increase its lipophilicity and reduce its negative charge, thus achieving enhanced delivery to the brain., Results: In this article, we have shown that conjugation of 12 nm GNPs with the amphipathic peptide CLPFFD increases the in vivo penetration of these particles to the rat brain. The C(GNP)-LPFFD conjugates showed a smaller negative charge and a greater hydrophobic character than citrate-capped GNPs of the same size. We administered intraperitoneal injections of citrate GNPs and C(GNP)-LPFFD in rats, and determined the gold content in the tissues by neutron activation. Compared with citrate GNPs, the C(GNP)-LPFFD conjugate improved the delivery to the brain, increasing the concentration of gold by fourfold, while simultaneously reducing its retention by the spleen 1 and 2 h after injection. At 24 h, the conjugate was partially cleared from the brain, and mainly accumulated in the liver. The C(GNP)-LPFFD did not alter the integrity of the blood-brain barrier, and had no effect on cell viability.

Portal hypertension is a major complication of cirrhosis that frequently leads to a neuropsychiatric disorder that affects cognition. We compared the performance of 18-month-old prehepatic portal hypertensive rats (PH) and 18-month-old normal rats (CO) in spatial short-term and reference memory tasks in the Morris water maze and in active avoidance task. The PH group showed worse spatial short-term memory than the CO group. Also, the PH group tended to perform worse than the CO group in the reference memory task, but it presented a correct acquisition of the active avoidance task. We assessed the brain metabolic activity of the animals by means of cytochrome c-oxidase (COx) histochemistry. We found that the PH group developed prefrontal dysfunction characterized by increased COx activity in this region compared to the CO group. Similar results were found in the medial mammillary nucleus and dentate gyrus, whereas the CA1 area, bed nucleus of the stria terminalis, and supramammillary nucleus showed lower COx activity in the PH group as compared to the CO group. We conclude that the 18-month-old portal hypertensive rats present spatial memory impairment without alteration of implicit learning. This deficit could be related to the alteration of the metabolic activity of the brain regions involved in the processing of spatial memories., (Copyright 2010 Elsevier Inc. All rights reserved.)

Several works have shown that males and females differ in the ability to learn spatial locations in mazes. In this study, we used the Morris water maze to assess the acquisition of a spatial working memory (WM) task in adult male and female Wistar rats. The task consisted of a paired sample procedure made up of two daily identical trials, sample and retention. To study the oxidative metabolic activity of some brain limbic system regions after the WM task, we applied the cytochrome oxidase (COx) histochemistry. In addition to the experimental groups, free swimming control groups and untrained naïve groups were added to explore the COx changes not specific to the learning process. Similar spatial performances were found between sexes as only one more sample and retention trials were needed in males to reduce the escape latencies significantly. Males showed decreased COx activity as compared to control groups in the medial prefrontal cortex (prelimbic and infralimbic regions) as well as in the lateral septum and dentate gyrus. Regarding females, an increase in COx activity was found in nucleus accumbens, ventral tegmental area and supramammillary region in relation to control groups. Overall, these findings suggest that the acquisition of the spatial WM task is mediated by different subsystems in a sex-dependent manner that points to the hippocampus as the central structure in males whereas other structures would be central in females.

Hepatic encephalopathy is a neurological complication observed in patients with liver disease. Subjects with hepatic encephalopathy can develop memory alterations. In order to investigate brain oxidative metabolism in an animal model of chronic cirrhosis and its modification after spatial working memory task, we determined the neural metabolic activity of several brain limbic system regions by cytochrome oxidase (COx) histochemistry and assessed the spatial working memory in the Morris water maze of rats with cirrhosis by administration of thioacetamide. This COx histochemistry was done in cirrhotic and control rats under basal conditions and after the spatial working memory task. The histochemical results showed differences in basal COx activity between control and cirrhotic rats in hippocampal and thalamic regions. In cirrhotic rats basal COx activity was increased in the CA1 and CA3 areas of the hippocampus and reduced in the anterodorsal and anteroventral thalamic nuclei. We found impaired spatial working memory in animals with cirrhosis. These animals showed absence of metabolic activation of the CA3 hippocampal subfield and the lateral mammillary nucleus and disturbance of COx activity in the medial mammillary nucleus and the anteroventral thalamus. These findings suggest that cirrhotic rats show spatial working memory deficits that could be related to the alteration of metabolic activity of neural regions thought to be involved in the processing of spatial memories.

The effects of antidepressant drugs on memory have been somewhat ignored, having been considered a mere side effect of these compounds. However, the memory impairment caused by several antidepressants could be considered to form part of their therapeutic effects. Amitriptyline is currently one of the most prescribed tricyclic antidepressants, and exerts marked anticholinergic and antihistaminergic effects. In this study, we evaluated the effects of inhibitory avoidance (IA) learning and acute administration of amitriptyline on brain oxidative metabolism. Brain oxidative metabolism was measured in several limbic regions using cytochrome oxidase (CO) quantitative histochemistry. Amitriptyline produced a clear impairment in the IA task. In animals exposed only to the apparatus, amitriptyline decreased CO activity in nine brain regions, without affecting the remaining regions. In animals that underwent the IA training phase, amitriptyline reduced CO activity in only three of these nine regions. In animals treated with saline, IA acquisition increased CO activity in the medial prefrontal cortex, the prelimbic cortex, and the medial mammillary body, and diminished it in the medial septum and the nucleus basalis of Meynert with respect to animals exposed only to the IA apparatus. In animals treated with amitriptyline, IA acquisition did not modify CO activity in any of these regions, but increased it in the anteromedial nucleus of the thalamus, the diagonal band of Broca, and the dentate gyrus. The results reveal a pattern of changes in brain oxidative metabolism induced by IA training in saline-treated animals that was clearly absent in animals submitted to the same behavioural training but treated with amitriptyline.

Although the hippocampus has been shown to be essential for spatial memory, the contribution of associated brain regions is not well established. Wistar rats were trained to find a hidden escape platform in the water maze during eight days. Following training, the oxidative metabolism in different brain regions was evaluated using cytochrome oxidase histochemistry. Metabolic activations were found in the prelimbic cortex, cornu ammonis (CA) 1 subfield of the dorsal hippocampus and the anterior thalamic nuclei, relative to yoked swim controls and naïve rats. In addition, many cross-correlations in brain metabolism were observed among the latter regions. These results support the implication of a hippocampal-prefrontal-thalamic system to spatial memory in rats.